Comprehensive analysis of insertion sequences within rRNA genes of CPR bacteria and biochemical characterization of a homing endonuclease encoded by these sequences.

The Candidate Phyla Radiation (CPR) represents an extensive bacterial clade comprising primarily uncultured lineages and is distinguished from other bacteria by a significant prevalence of insertion sequences (ISs) within their rRNA genes. However, our understanding of the taxonomic distribution and characteristics of these ISs remains limited. In this study, we used a comprehensive approach to systematically determine the nature of the rRNA ISs in CPR bacteria. The analysis of hundreds of rRNA gene sequences across 65 CPR phyla revealed that ISs are present in 48% of 16S rRNA genes and 82% of 23S rRNA genes, indicating a broad distribution across the CPR clade, with exceptions in the 16S and 23S rRNA genes of Candidatus (Ca.) Saccharibacteria and the 16S rRNA genes of Ca. Peregrinibacteria. Over half the ISs display a group-I-intron-like structure, whereas specific 16S rRNA gene ISs display features reminiscent of group II introns. The ISs frequently encode proteins with homing endonuclease (HE) domains, centered around the LAGLIDADG motif. The LAGLIDADG HE (LHE) proteins encoded by the rRNA ISs of CPR bacteria predominantly have a single-domain structure, deviating from the usual single- or double-domain configuration observed in typical prokaryotic LHEs. Experimental analysis of one LHE protein, I-ShaI from Ca. Shapirobacteria, confirmed that its endonuclease activity targets the DNA sequence of its insertion site, and chemical cross-linking experiments demonstrated its capacity to form homodimers. These results provide robust evidence supporting the hypothesis that the explosive proliferation of rRNA ISs in CPR bacteria was facilitated by mechanisms involving LHEs. IMPORTANCE: Insertion sequences (ISs) in rRNA genes are relatively limited and infrequent in most bacterial phyla. With a comprehensive bioinformatic analysis, we show that in CPR bacteria, these ISs occur in 48% of 16S rRNA genes and 82% of 23S rRNA genes. We also report the systematic and biochemical characterization of the LAGLIDADG homing endonucleases (LHEs) encoded by these ISs in the first such analysis of the CPR bacteria. This study significantly extends our understanding of the phylogenetic positions of rRNA ISs within CPR bacteria and the biochemical features of their LHEs.

Features of smaller ribosomes in candidate phyla radiation (CPR) bacteria revealed with a molecular evolutionary analysis.

The candidate phyla radiation (CPR) is a large bacterial group consisting mainly of uncultured lineages. They have small cells and small genomes, and they often lack ribosomal proteins uL1, bL9, and/or uL30, which are basically ubiquitous in non-CPR bacteria. Here, we comprehensively analyzed the genomic information on CPR bacteria and identified their unique properties. The distribution of protein lengths in CPR bacteria peaks at around 100-150 amino acids, whereas the position of the peak varies in the range of 100-300 amino acids in free-living non-CPR bacteria, and at around 100-200 amino acids in most symbiotic non-CPR bacteria. These results show that the proteins of CPR bacteria are smaller, on average, than those of free-living non-CPR bacteria, like those of symbiotic non-CPR bacteria. We found that ribosomal proteins bL28, uL29, bL32, and bL33 have been lost in CPR bacteria in a taxonomic lineage-specific manner. Moreover, the sequences of approximately half of all ribosomal proteins of CPR differ, in part, from those of non-CPR bacteria, with missing regions or specifically added regions. We also found that several regions in the 16S, 23S, and 5S rRNAs of CPR bacteria are lacking, which presumably caused the total predicted lengths of the three rRNAs of CPR bacteria to be smaller than those of non-CPR bacteria. The regions missing in the CPR ribosomal proteins and rRNAs are located near the surface of the ribosome, and some are close to one another. These observations suggest that ribosomes are smaller in CPR bacteria than those in free-living non-CPR bacteria, with simplified surface structures.

Complete Genome Sequence of Halophilic Deep-Sea Bacterium Halomonas axialensis Strain Althf1.

Halomonas axialensis is a halophilic bacterial species discovered near a deep-sea hydrothermal vent. Here, we report the first single closed genome sequence of the original strain, Halomonas axialensis strain Althf1. The genome was assembled by Nanopore sequencing and consisted of a single chromosome of 3.6 Mbp with 56.8% G+C content.